The field of the invention is inhibition of Smad2/3 signaling to promote regeneration of a lesioned CNS axon of a mature neuron.
The regeneration failure following injury to the adult mammalian CNS has been attributed to both the inhibitory extrinsic environment of the adult CNS, as well as the diminished intrinsic ability of mature axons to regenerate. Recent progress in examining the molecular mechanisms of inhibitory molecules associated with both CNS myelin and the glial scar has led to the development of various genetic and pharmacological means to block these inhibitory activities (Filbin, 2003; Harel and Strittmatter, 2006; Yiu and He, 2006; Schwab and Bartholdi, 1996; Silver and Miller, 2004). When applied to different in vivo injury models, however, these treatments have resulted in limited to no regeneration of lesioned axons (Case and Tessier-Lavigne, 2005; Thuret et al., 2006, Yiu and He, 2006). While it is possible that more comprehensive combinatorial treatments may further enhance the extent of axonal regeneration, a more likely possibility is that strategies that only target inhibitory signals are not sufficient to promote a significant level of regeneration. In fact, even a permissive environment, such as a sciatic nerve graft transplanted to the lesion site, only allows a small percentage of injured adult axons to regenerate into the graft (Aguayo et al., 1990; Schwab and Bartholdi, 1996). These results indicate that other mechanisms, such as those controlling the intrinsic axonal growth potential of neurons, may play important roles in axon regeneration.
Primary sensory neurons with cell bodies in trigeminal and dorsal root ganglia (DRG) have often been used as a model to study the mechanisms controlling the intrinsic growth ability of axons. These neurons possess two major branches stemming from a unipolar axon: a peripheral axon that innervates peripheral targets such as skin, viscera, and muscles, and a central axon that relays the information to the spinal cord or brain stem. In the adult, the peripheral and central branches differ dramatically with regard to their ability to regenerate after axotomy. While peripheral axons can readily regrow after lesioning, the injured central branch from the same DRG neuron may only sprout abortively but cannot regenerate. Interestingly, a conditioning lesion first made in the peripheral branch can dramatically increase the ability of the central axons to regenerate in the dorsal columns (Richardson and Issa, 1984; Richardson and Verge, 1986; Chong et al., 1996; Oudega et al., 1994; Neumann and Woolf, 1999; Neumann et al., 2005). In other words, the exuberant sprouting pattern of naive DRG neurons can be converted to efficient regenerative elongation by a priming peripheral nerve lesion (known as a “preconditioning lesion”). Importantly, these different regenerative competence states (before and after pre-conditioning lesion) can also be recapitulated in vitro. During the first day in culture, naive adult DRG neurons usually elaborate an “arborizing” growth pattern with compact, highly-branched arbors, while pre-conditioned cells exhibit an “elongating” phenotype with rapid extension of long, sparsely-branched axons (Smith and Skene, 1997; Bomze et al., 2001; Lankford et al., 1998; Liu and Snider, 2001; Neumann et al., 2005; Neumann and Woolf, 1999;). However, the molecular mechanism that triggers the loss of regenerative ability in mature neurons and how that may be reversed in pre-conditioned neurons remain unclear.
A possible clue to understanding the mechanisms controlling the regenerative ability of axons can be traced to the establishment of axonal connections during development. In general, when axons reach their targets, the motile growth cones of the developing axons progressively cease growing and transform over time into mature synaptic terminals with new functions and a distinct set of proteins (Hall and Sanes, 1993). For most neurons, this transformation involves the decreased expression of growth cone components (Basi et al., 1987; Caroni and Becker, 1992; Chu and Klymkowsky, 1989; Hoffman, 1989; Maness et al., 1988; Miller et al., 1989; Skene et al., 1986). It is conceivable then that the signals derived from presynaptic terminals and/or postsynaptic targets may instruct the neuronal somas to reprogram their growth states, thus leading to the loss of axonal regenerative ability in mature neurons. In this respect, it has been shown that a contact-dependent signal from amacrine cells innervating neonatal retinal ganglion neurons could trigger a loss of axonal growth ability (Goldberg et al., 2002). However, it remains unknown whether this mechanism may also apply to other types of neurons, and the identity of the molecular signal(s) that switch off the intrinsic regenerative capacity remains elusive.
In sensory neurons from DRGs, the time when axons reach their respective targets differs in different sub-groups. Cutaneous afferents appear in rat proximal hindlimb on embryonic days 14-15 (E14-15) and in the skin of toes at E16-17 (Mimics and Koerber, 1995a; Mimics and Koerber, 1995b). These fibers are functional soon afterwards, beginning at E18-19 (Fitzgerald, 1991; Saito, 1979). This is temporally correlated with the expression of different neurotransmitters such as calcitonin gene related peptide (CGRP) in DRG neurons innervating skin and viscera (Gibson et al., 1984; Hall et al., 1997; Lee et al., 1985a; Lee et al., 1985b), suggesting that target derived factors could act as retrograde signals to alter the program of neuronal differentiation. Interestingly, interruption of retrograde transport in the peripheral axons by transection, ligation, or local application of the microtubule-depolymerizing agent colchicine to the sciatic nerve, can enhance axonal growth ability when these neurons are cultured in vitro (Bomze et al., 2001; Lankford et al., 1998; Neumann and Woolf, 1999; Smith and Skene, 1997), consistent with the idea that signals emanating from peripheral targets also regulate the axonal growth ability of DRG neurons.
Several classes of molecules, such as neurotrophins, Wnts and TGFβ family members, have been implicated in regulating different aspects of neuronal differentiation and function via retrograde transport (reviewed by Ginty and Segal, 2002; Hippenmeyer et al., 2004; Howe and Mobley, 2005; Zweifel et al., 2005). A well-established example is the control of neuronal survival by the presence of limiting amounts of target-derived neurotrophic factors (Bibel and Barde, 2000; Campenot and MacInnis, 2004; Ginty and Segal, 2002; Zweifel et al., 2005). In addition, studies both in cell culture and in vivo have implicated TGFβ family members produced in skin, notably activin A, as inducers of CGRP expression in DRG neurons (Ai et al., 1999; Hall et al., 1997; Hall et al., 2001 McMahon et al., 1989). TGFβ family members, which include activins, TGFβs, and BMPs among others, bind to type I and type II cell surface receptors (Shi and Massague, 2003). A major class of downstream mediators known as the Smad family of signal transducing proteins could then relay the activated signal from the cell membrane of axonal terminals to the nucleus via retrograde transport. In the nucleus, Smads collaborate with other co-activators and repressors to trigger transcriptional responses that are specific for different biological processes (Attisano and Wrana, 2002; Derynck and Zhang, 2003; Massague et al., 2005).
TGFβ members and their receptors are expressed in peripheral targets and sensory neurons not only during development but also in adulthood (Ai et al., 1999; Rogister et al., 1993; Stark et al., 2001). Moon and Fawcett (Eur J Neurosci. (2001) 14:1667-77) report a reduction in CNS scar formation without concomitant increase in axon regeneration following treatment of adult rat brain with a combination of antibodies to TGFβ1 and TGFβ2.
In studies with embryonic Xenopus explants, both truncated activin type II receptor and follistatin (a protein derived from the embryonic Spemann Organizer) block activin signaling and neuralize cells that would otherwise become epidermal cells (Hemmati-Brivanlou et al. Cell (1994) 77:273-281; Cell (1994) 77:283-295). These Hemmati-Brivanlou et al. papers only address differentiation of embryonic cells, and do not mention, discuss, nor have any relevance to differentiated, adult cell types, such as post-mitotic, differentiated adult neurons.
Hemmati-Brivanlou et al. submitted these same data in a patent application (now, U.S. Pat. No. 6,686,198) which they accurately abstract as relating to differentiation of embryonic cells: “The subject method stems from the unexpected finding that, contrary to traditional understanding of neural induction, the default fate of ectodermal tissue is neuronal rather than mesodermal and/or epidermal. In particular, it has been discovered that preventing or antagonizing a signaling pathway in a cell for a growth factor of the TGFβ. family can result in neuronal differentiation of that cell.”
However, unlike their teachings to their peers skilled in the art (Hemmati-Brivanlou et al, supra), their patent disclosure erects a remarkable proposal: “The ability of follistatin to regulate neuronal differentiation not only during development of the nervous system but also presumably in the adult state . . . indicates that NAs can be reasonably expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells.” (U.S. Pat. No. 6,686,198; Col. 13, lines 35-10). Other than a “presumably” the patent offers no support whatsoever for this scientifically untenable proposal. As noted above, the only documentation presented relates to differentiation of embryonic Xenopus cells, which evidence those skilled in this art recognize as not probative of any effect on post-mitotic, terminally-differentiated cells like adult neurons.